Research this past year focussed on the molecular characterization of glutamate receptors and their function in the auditory system. Sequence analysis of cochlear PCR products showed, in addition to GluR1-4 subunits, the presence of a novel form of GluR3 in the cochlea that is not found in brain. This new form is identical to GiuR3 except it is missing a 99 bp sequence encoding 33 amino acids between the third and fourth putative transmembrane regions, an area of the molecule which contains two consensus protein kinase c phosphorylation sites. MRNAS for GluR-4 are expressed in the cochlear nucleus at all stages of development, but GluR1 expression appears to be more extensive in the developing animal, compared to the adult. Subunit-specific antibodies were used for immunocytochemical analysis in the brain and cultured hippocampal neurons. In the cochlear nucleus, antibodies to GluR2/3 showed the widest distribution with moderate to dense staining of all major types of neurons. The most distinctive labeling was seen with antibodies to GiuR1, with dense staining restricted to the superficial dorsal cochlear nucleus. GluR1-4 immunoreactivity was present in cell bodies and dendrites, but not axons, of cultured hippocampal neurons. Using intact living neurons, receptors on the surface of the neurons were not labeled with antibodies made to the C-terminus suggesting that this region of the molecule may not be exposed on the external membrane as expected. No selective targeting of individual GluR subunits or their MRNAS was seen. An antibody specific for KA2 showed moderate to dense staining in a number of structures including the hippocampus, reticulothalamic nucleus, and dorsal root ganglia. At the ultrastructural level staining was very dense at the postsynaptic densities and moderate in adjacent dendritoplasm at synapses of the cerebral cortex and hippocampus. This included mossy fiber terminals on apical dendrites of the CA3 region, a population of synapses in which we have shown staining with antibodies for GiuR1-4 subunits. Using the Xenopus oocyte in vitro translation system, we showed that NAAG acts as an agonist on homomeric NMDA receptors (NMDAR). NAAG induced current responses in oocytes expressing NMDA receptors, while it had little or no effect on oocytes expressing other glutamate receptors. Our further analysis of novel members of the dipeptidyl aminopeptidase family led to the identification of human homologues. The original form did not have enzymatic activity; however, mutation of a single amino acid led to enzymatic activity.